Prevention of protein disulfide bond reduction

Abstract

Disclosed are methods for testing the presence and/or activity of glutathione system components and thioredoxin system components during the manufacturing process of disulfide bond-containing proteins. Also disclosed are methods for mitigating reduction of disulfide bonds during the manufacturing process, and for lowering the reduction potential of disulfide bond-containing proteins. Provided are compositions, kits, and methods for mitigating reduction and diminishing reduction potential of disulfide bond-containing proteins during protein manufacturing processes.

What is claimed is: 
     
         1 . A method for increasing the yield of an intact disulfide bond-containing antibody or antigen-binding fragment thereof in a cell culture or solution, comprising manufacturing the disulfide bond-containing antibody or antigen-binding fragment thereof in the presence of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or both a glutathione reductase inhibitor and a thioredoxin reductase inhibitor,
 detecting the presence of glutathione reductase and/or thioredoxin reductase in the culture or the solution containing the disulfide bond-containing antibody or antigen-binding fragment thereof, and   adding a glutathione reductase and/or thioredoxin reductase inhibitor to mitigate reduction of the disulfide bond-containing antibody or antigen-binding fragment thereof;   whereby the amount of intact disulfide bond-containing antibody or antigen-binding fragment thereof is higher in the cell culture or solution manufactured in the presence of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or a glutathione reductase inhibitor and a thioredoxin reductase inhibitor as compared to the amount of intact disulfide bond-containing antibody or antigen-binding fragment thereof in a cell culture or solution not manufactured in the presence of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or a glutathione reductase inhibitor and a thioredoxin reductase inhibitor.   
     
     
         2 . The method of  claim 1  where increasing the yield of an intact disulfide bond-containing antibody or antigen binding fragment thereof in a cell culture or solution is controlled via an online cell culture redox potential control system, wherein the online measurement of the cell culture redox potential is determined to increase the concentration of a glutathione reductase inhibitor, a thioredoxin reductase inhibitor, or a glutathione reductase inhibitor and a thioredoxin reductase inhibitor in response to a decrease in the cell culture redox potential. 
     
     
         3 . The method of  claim 1  wherein an increase of the yield of an intact disulfide bond-containing antibody or antigen-binding fragment thereof in a cell culture or solution is monitored online via measurement of the cell culture redox potential throughout the manufacturing process to prevent unnecessary over-addition of the glutathione reductase inhibitor, thioredoxin reductase inhibitor, or glutathione reductase inhibitor and thioredoxin reductase inhibitor in the final process titer. 
     
     
         4 . The method of  claim 1 , further comprising determining the activity of the glutathione reductase and/or thioredoxin reductase in the culture or the solution during the manufacturing process and after a first addition of a glutathione reductase and/or a thioredoxin reductase inhibitor to continue to monitor and mitigate reduction. 
     
     
         5 . The method of  claim 1 or claim 4 , wherein determining the activity of the glutathione reductase and/or thioredoxin reductase in the culture or the solution comprises:
 adding 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to a sample obtained during the manufacturing process;   adding at least one of thioredoxin reductase inhibitor and glutathione reductase inhibitor to a portion of the sample containing DTNB; and   monitoring reduction of DTNB at a wavelength of 412 nm in the sample;   
       wherein a higher reduction of DTNB in the samples without at least one of thioredoxin reductase inhibitor and glutathione reductase inhibitor, indicates the activity of thioredoxin reductase and/or glutathione reductase. 
     
     
         6 . The method of  claim 5 , wherein NADPH, oxidized glutathione, and buffer are added to the sample prior to monitoring reduction. 
     
     
         7 . A method for increasing the yield of an intact disulfide bond-containing antibody or antigen-binding fragment thereof or diminishing the reduction potential of the disulfide containing antibody or antigen-binding fragment thereof in a cell culture or fermentation process comprising maintaining the extracellular cystine levels above 0 and/or maintaining the extracellular Cu levels above 0 and/or adding EDTA to the bioreactor during the cell culture or fermentation process or to the solution containing the disulfide bond-containing antibody or antigen-binding fragment thereof; wherein maintaining the extracellular cystine levels above 0 during the cell culture or fermentation process prevents disulfide bond reduction in the disulfide bond-containing antibody or antigen-binding fragment thereof. 
     
     
         8 . The method of  claim 7 , wherein the intact disulfide bond-containing antibody or antigen-binding fragment thereof is released into the cell culture fluid (CCF). 
     
     
         9 . The method of  claim 8 , wherein the cell culture or fermentation process comprises maintaining the cells in CCF for at least 2 days. 
     
     
         10 . The method of  claim 9 , wherein the cell culture process is a mammalian or insect cell culture process, and the fermentation process is a bacterial, yeast, or fungi fermentation process. 
     
     
         11 . A method for improving the stability of a purified disulfide bond-containing antibody or antigen-binding fragment thereof, comprising using a manufacturing process that mitigates reduction or reduction potential of the antibody or antigen-binding fragment thereof during the manufacturing process, thereby resulting in lower aggregate formation during the purification process, wherein the manufacturing process comprises a cell culture phase, a harvest phase, at least one hold phase, a purification phase, or any combination thereof. 
     
     
         12 . The method of  claim 11 , where the minimization of disulfide bond reduction and/or disulfide bond reduction potential and/or free thiols is achieved by increasing Zn2+, Mn2+, Fe3+, Cu2+, Se2+, cystine, dissolved oxygen, beta mercaptoethanol, or glutathione, or a combination thereof during any step of the manufacturing process, as long as the cell culture redox potential is maintained above a threshold value of −55 mV or −70 mV. 
     
     
         13 . A method for increasing the yield of an intact disulfide bond-containing antibody or antigen-binding fragment thereof in a cell culture or solution, comprising manufacturing the disulfide bond-containing antibody or antigen-binding fragment thereof using a process that mitigates reduction or reduction potential of the antibody or antigen-binding fragment thereof by increasing the concentration of Zn2+, Mn2+, Fe3+, Cu2+, Se2+, cystine, beta-mercapto ethanol, glutathione, or dissolved oxygen during manufacturing processes, comprising detecting the redox potential in the culture or solution containing the disulfide bond-containing antibody or antigen-binding fragment thereof, and adding of Zn2+, Mn2+, Fe3+, Cu2+, Se2+, cystine, beta-mercaptoethanol, glutathione, or dissolved oxygen to mitigate reduction of the disulfide bond-containing antibody or antigen-binding fragment thereof; wherein the cell culture redox potential is maintained above a threshold value of −55 mV or −70 mV, thereby increasing the yield of the intact disulfide bond-containing antibody or antigen-binding fragment.